Project Summary Recent developments in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for illuminating cellular structures at the nanoscale. It is now feasible to visualize and quantify the spatial organization of proteins and other macromolecules that enable cells to sense and respond to their environment. However, these efforts are restricted by the shortage of methods for attaching FM-, EM-, and CLEM-compatible reporter chemistries to target proteins. We will address this limitation by developing a new technology for labeling and imaging multiple cellular proteins at once. Specifically, we propose to develop a set of heterodimeric coiled-coil tags that will allow specific protein labeling for FM and EM. Our new versatile interacting peptide (VIP) tags will be protein specific and cell compatible. Briefly, one coil (the ?tag?) will be genetically-encoded as a fusion to a protein of interest. After expression, the tagged protein will be subsequently labeled via heterodimer formation with a high affinity (KD < 5 nM) ?probe peptide?. We identified a set of coils that will self-sort into specific pairs, which will enable up to four proteins to be labeled and imaged simultaneously. The reporter can be bright, photostable fluorophores for FM or electron-dense nanoparticles for EM. In other words, VIP tags are modular, enabling end-users to alternate between state-of-the art FM and EM imaging platforms. We will engineer VIP tags to achieve the high labeling efficiency needed for quantitative analysis of multi-protein interactions. We will develop and validate our technology by investigating the iron-uptake machinery in cells. We will use our technology to determine the differential protein trafficking and interactions of transferrin receptors 1 and 2 (TfR1 and TfR2). TfR1 is a well-studied transmembrane receptor and an ideal target for validating our technology. Our studies of TfR2 will reveal new information on the sub-cellular distribution and trafficking of this recently discovered iron-sensing receptor. We propose to complete two Aims. Aim 1. Develop and validate a set of VIP tags for imaging proteins by FM. Aim 2. Use VIP tags for tracking receptor localization and multi-protein interactions by EM. We believe that the VIP tags, once fully developed and validated, will be an ideal technology for investigating the cellular organization of proteins with nanoscale precision. Furthermore, we believe this new technology has broad utility for imaging cellular processes related to human health and disease.